Structural and functional insights into the bona fide catalytic state of Streptococcus pyogenes Cas9 HNH nuclease domain

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Abstract

The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (SpyCas9), along with a programmable single-guide RNA (sgRNA), has been exploited as a significant genome-editing tool. Despite the recent advances in determining the SpyCas9 structures and DNA cleavage mechanism, the cleavage-competent conformation of the catalytic HNH nuclease domain of SpyCas9 remains largely elusive and debatable. By integrating computational and experimental approaches, we unveiled and validated the activated Cas9-sgRNA-DNA ternary complex in which the HNH domain is neatly poised for cleaving the target DNA strand. In this catalysis model, the HNH employs the catalytic triad of D839-H840-N863 for cleavage catalysis, rather than previously implicated D839-H840-D861, D839-H840-N854, D837-D839-H840, or D839-H840-D861-N863. Our study contributes critical information to defining the catalytic conformation of the HNH domain and advances the knowledge about the conformational activation underlying Cas9-mediated DNA cleavage.

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The data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 and 2.

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Zhicheng Zuo

College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, China

Competing interests
The authors declare that no competing interests exist.
Ashwini Zolekar

Department of Pharmaceutical Sciences, University of North Texas Health Science Center, Fort Worth, United States

Competing interests
The authors declare that no competing interests exist.
Kesavan Babu

Department of Chemistry and Biochemistry, University of Oklahoma, Norman, United States

Competing interests
The authors declare that no competing interests exist.
Victor JT Lin

Department of Pharmaceutical Sciences, University of North Texas Health Science Center, Fort Worth, United States

Competing interests
The authors declare that no competing interests exist.
Hamed S Hayatshahi

Department of Pharmaceutical Sciences, University of North Texas Health Science Center, Fort Worth, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8639-7130
Rakhi Rajan

Department of Chemistry and Biochemistry, University of Oklahoma, Norman, United States

Competing interests
The authors declare that no competing interests exist.
Yu-Chieh Wang

Department of Pharmaceutical Sciences, University of North Texas Health Science Center, Fort Worth, United States

For correspondence
yu-chieh.wang@unthsc.edu

Competing interests
The authors declare that no competing interests exist.
Jin Liu

Department of Pharmaceutical Sciences, University of North Texas Health Science Center, Fort Worth, United States

For correspondence
jin.liu@unthsc.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1067-4063

Funding

Shanghai Municipal Education Commission (Program for Professor of Special Appointment at Shanghai Institutions of Higher Learning)

Zhicheng Zuo

National Science Foundation (MCB-1716423)

Rakhi Rajan

National Institute of General Medical Sciences (P20GM103640)

Rakhi Rajan

University of North Texas Health Science Center (Start-up Fund and Faculty Pilot Grant)

Yu-Chieh Wang

University of North Texas Health Science Center (Start-up Fund and Basic Research Seed Grant)

Jin Liu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.